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Method for labelling and tracing uterine epithelial cell-derived exosome by using DiI dye

A technology for epithelial cells and exosomes, applied in the field of DiI dye labeling and tracing of exosomes derived from uterine epithelial cells, can solve the problem of affecting the function of exosomes, destroying the membrane structure of exosomes, changing the composition of exosomes, etc. question

Inactive Publication Date: 2019-12-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] So far, there have been a variety of methods for labeling exosomes, but there are certain defects, mainly in the destruction of the membrane structure of exosomes, changing the composition of exosomes, affecting the function of exosomes, etc.

Method used

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  • Method for labelling and tracing uterine epithelial cell-derived exosome by using DiI dye
  • Method for labelling and tracing uterine epithelial cell-derived exosome by using DiI dye
  • Method for labelling and tracing uterine epithelial cell-derived exosome by using DiI dye

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Embodiment 1

[0035] Experimental steps:

[0036] 1. Exosomes were extracted from the supernatant of ishikawa (from Shanghai Cell Bank of Chinese Academy of Sciences) and HEC-1-A (Shanghai Cell Bank of Chinese Academy of Sciences) by ultracentrifugation. The brief description is as follows: 150ml supernatant was centrifuged at 300g for 10min, 2000g for 20min, 10000g, 30min, the obtained supernatant was passed through a 0.22um filter membrane, centrifuged twice at 120000g, all the above centrifugations were carried out at 4°C, a total of about 80ug exosomes were obtained, and incubated with 20uM DiI dye (60ul) for 30min at 37°C;

[0037] 2. Centrifuge at 120,000 g at 4°C for 70 min to remove excess dye and precipitate exosomes. The obtained exosomes are resuspended in 200ulPBS, and the supernatant (that is, DiI dye alone) is used as a control, and added to HTR8 cells in 1% serum medium. Incubate at 37°C for 4-6 hours;

[0038] 3. Take out the cell culture plate and wash it 3 times with PBS,...

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Abstract

The invention aims at providing a method for labelling and tracing a uterine epithelial cell-derived exosome by using a DiI dye without changing the structure and components of the exosome or affecting biological functions of the exosome. The method comprises the steps of adding an exosome into 10-30nM of DiI dye for incubation at 37 DEG C for 30min, carrying out centrifuging and removing excessive dye; and resuspending the obtained exosome by using PBS and obtaining the labelled exosome. The method has the main beneficial effects that the structure and components of the exosome are changed, the biological functions of the exosome are not affected, the process of uptaking the uterine epithelial cell-derived exosome through embryonic trophoblast cells can be accurately traced and further study on the functions of the exosome is facilitated.

Description

[0001] (1) Technical field [0002] The invention relates to a method for labeling and tracing exosomes derived from uterine epithelial cells with DiI dye. [0003] (2) Background technology [0004] DiI is DiIC18(3), the full name is 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, the molecular formula is C 59 h 97 ClN 2 o 4 (Structure shown in formula (I)), molecular weight is 933.88, and CAS number is 41085-99-8. DiI can be dissolved in absolute ethanol, DMSO and DMF, wherein the solubility in DMSO is greater than 10 mg / ml. It is one of the most commonly used fluorescent probes for cell membranes, showing orange-red fluorescence. DiI is a lipophilic membrane dye. After entering the cell membrane, it can diffuse laterally to gradually stain the cell membrane of the entire cell. For the excitation and emission spectra of DiI combined with the phospholipid bilayer membrane, see figure 1 . Before DiI enters the cell membrane, the fluorescence is very w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N21/64
CPCG01N1/28G01N21/6428G01N2021/6439
Inventor 谭强施爽褚玲娜邱寒峰王争光
Owner ZHEJIANG UNIV